erbB3 Is an Active Tyrosine Kinase Capable of Homo- and Heterointeractions

Often considered to be a “dead” kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate, and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. erbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally small amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. erbB3 homo- and heterodimerization events were captured in real time on live cells using single-particle tracking of quantum dot probes bound to ligand or hemagglutinin tags on recombinant receptors.     

Cell type dependent endocytic internalization of ErbB2 with an artificial peptide ligand that binds to ErbB2

ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed.     

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.     

Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: local stimulation using magnetic microspheres as assessed by quantitative digital microscopy.

BACKGROUND: ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. METHODS: Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed stimulation of cells. We used EGF- and trastuzumab-covered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. RESULTS: On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2-mYFP F4 cell line), EGF-coupled-microspheres activated erbB1 and transactivated erbB2-mYFP. In two other cell lines with comparable erbB2 expression but lower levels of erbB1, EGF microspheres transactivated erbB2 less efficiently. Trastuzumab in solution activated erbB2 on A4-erbB2-mYFP and the trastuzumab sensitive SKBR-3 cells, but only negligibly on the resistant JIMT-1 cells that showed a 10 times higher K(d) for the antibody. Nevertheless, pronounced erbB2 activation and tyrosine phosphorylation could be detected after stimulation with trastuzumab-coupled microspheres in all cell lines, although transactivation of erbB1 was negligible. Receptor phosphorylation was restricted to the immediate proximity of the microspheres, i.e., receptor clusters external to these locations remained inactive. CONCLUSION: ErbB1 ligand and erbB2 specific antibody attached to magnetic microspheres are efficient tools in assessing erbB activation, localized signal propagation, and erbB heterodimer formation. Trastuzumab coupled to microspheres is more efficient at accessing erbB2 and activating it than trastuzumab in solution.     

erbB-2与胃癌细胞恶性表型及其增殖调控的关系

应用基因重组、基因转染、Ｓｏｕｔｈｅｒｎ杂交、Ｎｏｒｔｈｅｒｎ杂交、细胞生长曲线测定及裸鼠成瘤观察了反义ｅｒｂＢ－２逆转录病毒重组载体的转染对人胃癌细胞中ｅｒｂＢ－２过度表达的影响及其对ＥＧＦ的反应性．研究结果显示,ｅｒｂＢ－２的表达被其反义重组子特异抑制,伴有细胞增殖能力的下降及致瘤性的下降;在ＥＧＦ的刺激下,ｅｒｂＢ－２高表达肿瘤细胞生长速度提高的幅度显著大于ｅｒｂＢ－２反义重组载体转染细胞;ＥＧＦ促细胞增殖及促基因表达的功能在ｅｒｂＢ－２反义重组子转染后受到抑制,提示ｅｒｂＢ－２在细胞增殖调控中具有重要功能．     

Disabling receptor ensembles with rationally designed interface peptidomimetics

Members of the erbB family receptor tyrosine kinases (erbB1, erbB2, erbB3, and erbB4) are overexpressed in a variety of human cancers and represent important targets for the structure-based drug design. Homo- and heterodimerization (oligomerization) of the erbB receptors are known to be critical events for receptor signaling. To block receptor self-associations, we have designed a series of peptides derived from potential dimerization surfaces in the extracellular subdomain IV of the erbB receptors (erbB peptides). In surface plasmon resonance (BIAcore) studies, the designed peptides have been shown to selectively bind to the erbB receptor ectodomains and isolated subdomain IV of erbB2 with submicromolar affinities and to inhibit heregulin-induced interactions of erbB3 with different erbB receptors. A dose-dependent inhibition of native erbB receptor dimerization by the erbB peptides has been observed in 32D cell lines transfected with different combinations of erbB receptors. The peptides effectively inhibited growth of two types of transformed cells overexpressing different erbB receptors, T6-17 and 32D, in standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell viability assays. The study identifies distinct loops within the membrane-proximal part of the subdomain IV as potential receptor-receptor interaction sites for the erbB receptors and demonstrates the possibility of disabling receptor activity by structure-based targeting of the dimerization interfaces. Molecular models for possible arrangement of the erbB1.EGF complex, consistent with the involvement of subdomain IV in inter-receptor interactions, are proposed. Small dimerization inhibitors described herein can be useful as probes to elucidate different erbB signaling pathways and may be developed as therapeutic agents.     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

ErbB2 down-regulates microRNA-205 in breast cancer

Gene amplification and protein overexpression of erbB2 (Her2/neu) has been observed in approximately 20–30% of breast cancers. ErbB2-positive breast cancer is tend to be more aggressive than other types of breast cancer and therefore further investigation on the signaling pathways of erbB2 is needed for the therapeutic target for breast cancer treatment. Here we report that microRNA-205 (miR-205), a molecule also reported to be associated with breast cancer, is negatively regulated by erbB2 overexpression. Breast epithelial cells exogenously overexpressed with erbB2 decreased the expression of miR-205, whereas increased the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase 6 (CDK6). The decreased expression of miR-205 slightly increased by the transfection of erbB2 siRNA into the erbB2-overexpressing breast cancer epithelial cells. Overexpression of erbB2 enabled breast epithelial cells to grow anchorage-independently in soft agar, and the transfection of the precursor of miR-205 into the cells leaded to the decrease in the ability to grow in soft agar. These results suggest that down-regulation of miR-205 in erbB2-overexpressing breast epithelial cells is essential for erbB2-induced tumorigenesis, and miR-205 may have the potential to be a novel important alternative therapeutic target for erbB2-positive breast cancer.     

Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.     

Adult rat otic placode-derived neurons and sensory epithelium express all four erbB receptors: a role in regulating vestibular ganglion neuron viability.

The erbB receptor family consists of erbB1/epidermal growth factor receptor, erbB2/neu, erbB3, and erbB4, all of which have been implicated in cell proliferation, differentiation, and survival in several tissues. In the nervous system, these family members can function in a trophic capacity for certain subpopulations of neurons and some types of non-neuronal cells. Vestibular sensory epithelial cells and vestibular ganglion neurons are derived from ectodermal otic placode and are essential components of the peripheral vestibular system, the sensory system for balance. Recent studies in mammals suggest that certain ligands of the epidermal growth factor receptor can induce proliferation of vestibular sensory epithelial cells. We now show that vestibular ganglion neurons and vestibular sensory epithelial cells express all four erbB receptors in adult rats. Cultured vestibular ganglion neurons also expressed all four erbB family members and were therefore used to analyze the effects of modulating erbB signaling on differentiated vestibular ganglion neurons. Transforming growth factor-alpha (a ligand for epidermal growth factor receptor) and sensory and motor neuron-derived factor (a ligand for erbB3 and erbB4) promoted vestibular ganglion neuron viability, whereas epidermal growth factor (another ligand for epidermal growth factor receptor) did not. Glial growth factor 2 (another ligand for erbB3 and erbB4) and an antibody that blocks erbB2/neu-mediated signaling inhibited vestibular ganglion neuron viability. Collectively, these observations indicate that erbB signaling regulates the viability of differentiated otic placode-derived cells in mammals and suggest that exogenous modulation of erbB signaling in peripheral vestibular tissues may prove therapeutically useful in peripheral vestibular disorders.     

The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes

Development of oligodendrocytes and the generation of myelin internodes within the spinal cord depends on regional signals derived from the notochord and axonally derived signals. Neuregulin 1 (NRG)-1, localized in the floor plate as well as in motor and sensory neurons, is necessary for normal oligodendrocyte development. Oligodendrocytes respond to NRGs by activating members of the erbB receptor tyrosine kinase family. Here, we show that erbB2 is not necessary for the early stages of oligodendrocyte precursor development, but is essential for proligodendroblasts to differentiate into galactosylcerebroside-positive (GalC+) oligodendrocytes. In the presence of erbB2, oligodendrocyte development is normal. In the absence of erbB2 (erbB2-/-), however, oligodendrocyte development is halted at the proligodendroblast stage with a >10-fold reduction in the number of GalC+ oligodendrocytes. ErbB2 appears to function in the transition of proligodendroblast to oligodendrocyte by transducing a terminal differentiation signal, since there is no evidence of increased oligodendrocyte death in the absence of erbB2. Furthermore, known survival signals for oligodendrocytes increase oligodendrocyte numbers in the presence of erbB2, but fail to do so in the absence of erbB2. Of the erbB2-/- oligodendrocytes that do differentiate, all fail to ensheath neurites. These data suggest that erbB2 is required for the terminal differentiation of oligodendrocytes and for development of myelin.     

Metformin inhibits breast cancer cell growth,colony formation and induces cell cycle arrest in vitro

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G1 checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27kip1 or p21waf1. It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.     

Colonic epithelial expression of ErbB2 is required for postnatal maintenance of the enteric nervous system

We utilized the Cre-LoxP system to establish erbB2 conditional mutant mice in order to investigate the role of erbB2 in postnatal development of the enteric nervous system. The erbB2/nestin-Cre conditional mutants exhibit retarded growth, distended colons, and premature death, resembling human Hirschsprung's disease. Enteric neurons and glia are present at birth in the colon of erbB2/nestin-Cre mutants; however, a marked loss of multiple classes of enteric neurons and glia occurs by 3 weeks of age. Furthermore, we demonstrate that the requirement for erbB2 in maintaining the enteric nervous system is not cell autonomous, but rather erbB2 signaling in the colonic epithelia is required for the postnatal survival of enteric neurons and glia.     

Prolonged EGFR signaling by ERBB2-mediated sequestration at the plasma membrane

We have analyzed the spatial-temporal regulation of epidermal growth factor receptor (EGFR) phosphorylation by the orphan erbB2 receptor. It is shown that EGFR association with erbB2 is sufficient to prolong and enhance the net phosphorylation of EGFR, independent of the kinase activity of erbB2. This enhanced EGFR signaling was rather caused by erbB2-mediated retention of phosphorylated EGFR at the plasma membrane (PM), thereby preventing EGFR dephosphorylation and signal termination by endomembrane-bound protein tyrosine phosphatases (PTPs). EGF-induced EGFR internalization was indeed blocked in the presence of high levels of erbB2 or if cbl binding of EGFR was impaired. This erbB2-mediated blockage of the entry of activated EGFR into clathrin-coated vesicles could be alleviated by antibody-mediated disruption of the interaction between EGFR and erbB2. These results identify erbB2-mediated dominant trapping of phosphorylated EGFR at the PM as a mechanism that prolongs EGFR signaling, by sequestration of activated EGFR away from intracellular sites of high PTP activity.     

Expression of the Activated p185Tyrosine Kinase in Human Epithelial Cells Leads to MAP Kinase Activation but Does Not Confer Oncogenicity

Amplification of thec-erbB2gene and overexpression of p185^erbB2is found in approximately one-third of primary breast and ovarian cancers and also in some colon carcinomas. Moreover, a single point mutation inerbB2(V 664 E)confers transforming potential to erbB2 in NIH3T3 cells, even when expressed at low levels. To examine the transformation potential oferbB2orerbB2(V-E)in colon epithelial cells, we have transfected a nontumorigenic clone of SW 613-S cells with either wild-type p185^erbB2or mutated p185^erbB2(V-E). In contrast to p185^erbB2, p185^erbB2(V-E)associated constitutively with members of the Shc protein family, leading to phosphorylation of Shc and to stimulation of mitogen-activated protein kinase (MAP kinase). However, constitutive activation of MAP kinase activation in p185^erbB2(V-E)expressing cells did not result in a tumorigenic phenotype. In addition, p185^erbB2(V-E)expressing cells displayed a reduced ability to grow in soft agar compared to the parental cell line. In contrast these transfected cells were able to grow in three-dimensional collagen gels, whereas parental cells were not. Thus, expression oferbB2(V-E)in SW 613-S cells induced multiple changes in intracellular signaling and in growth requirement phenotype, particularly in response to the extracellular environment.     

Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.     

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors.     

The Constitutive Activity of Epidermal Growth Factor Receptor vIII Leads to Activation and Differential Trafficking of Wild-type Epidermal Growth Factor Receptor and erbB2

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII''s activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII''s activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. (J Histochem Cytochem 58:529–541, 2010)